In-vitro Characterization of an L-Kynurenine-Responsive Transcription Regulator of the Oxidative Tryptophan Degradation Pathway in Burkholderia xenovorans

Abstract

To study the transcriptional regulation of oxidative tryptophan degradation in Burkholderia, we used comparative genomics that focused on the operon containing the genes annotated as kynA, kynU and kynB. In all sequenced β-proteobacteria to-date, including Burkholderia, Ralstonia, Collimonas, and Cupriavidus species, there is a conserved AsnC/Lrp family transcriptional regulator (TR) gene located upstream and in the opposite strand of the operon encoding for the oxidative tryptophan degradation genes. In Burkholderia xenovorans the TR is Bxe_A0736. GST-Bxe_A0736 binds L-kynurenine with greater affinity and specificity than any other amino acid or tryptophan degradation product with a dissociation constant of ~82 ± 11 μM. DNaseI footprinting suggested that Bxe_A0736 protects a set of four degenerate, palindromic sequences within the intergenic region between Bxe_A0735 (kynB) and Bxe_A0736. The optimal consensus sequence obtained by analysis for these sites, ATATTCCGAATAT, closely resembles the sequence obtained with a protein binding microarray. Under our fluorescence anisotropy experimental conditions, 1 mM L-kynurenine increased the affinity of Bxe_A0736 for a portion of its promoter region. Our results are consistent with Bxe_A0736 acting as the TR that promotes the transcription of the oxidative tryptophan degradation genes in the presence of L-kynurenine while inhibiting the transcription of its own gene.

Automated genomic context analysis and experimental validation platform for discovery of prokaryote transcriptional regulator functions

“…Figure 2 presents the graphic user interface (GUI) for Function Discovery V1.0 showing the parameters used for the validation test runs. For nine TRs, including BetI [EMBL: ABE34374] [ 22 ], CatR [EMBL: ABE30799] [ 23 ], CynR [EMBL: ABE29438] [ 24 ], CysB [EMBL: ABE30507] [ 25 ], GlpR [EMBL: ABE32291] [ 26 ], HpaR [EMBL: ABE33958] [ 27 ], KynR [EMBL: ABE32198] [ 28 , 29 ], HutC [EMBL: ABE30031] [ 30 , 31 ] and RcoM [EMBL: ABE30826] [ 32 ], the pathways known to be regulated by the TRs were identified in the first hit in the Over-represented Pathway Summary (Additional files 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 and 10 ); in each case the TR effector molecules are metabolic intermediates in the pathway they regulate. The exception was ModE which required a closer analysis of the Function Discovery V1.0 report (Additional file 11 ).…”

“…It does not restrict our investigation to a single organism in which gene clustering around the target TR might not have existed because of rearrangements, insertions or deletions of genetic material or other events. Recently, we reported the characterization of three TRs of unknown function in which we used a manual version of our approach to predict functionality and identify potential effector metabolites [ 29 , 38 , 39 ]. Our automated approach, Function Discovery V1.0 has also been effective in finding conserved genes clustered near our target genes and for ten randomly chosen TRs that have known effectors and functions (Table 1 ).…”